Quantitative mRNA expression analysis of neurotrophin‐receptor TrkC and oncogene c‐MYC from formalin‐fixed,paraffin‐embedded primitive neuroectodermal tumor samples

Most recent studies analyzing candidate biological prognostic factors (including neurotrophin receptor TrkC and proto‐oncogene c‐MYC) in childhood primitive neuroectodermal brain tumors (PNET) are limited by small patient numbers due to dependence on fresh‐frozen tumor material. In contrast, large archives of formalin‐fixed, paraffin‐embedded PNET samples exist from homogeneously treated patients. The ability of real‐time RT‐PCR to assay very small mRNA fragments makes this assay amenable to studies where the RNA is moderately or even highly degraded. We have optimized RNA isolation from archive PNET samples and found that TrkC and c‐MYC mRNA measurements significantly correlated with those obtained from matching fresh‐frozen tissues. Exploitation of already existing archives of formalin‐fixed paraffin‐embedded PNET samples may accelerate the building of better stratification systems for PNET patients.     

Cocaine inhibition of neuronal differentiation in NGF-induced PC12 cells is independent of ras signaling

In utero exposure to cocaine may result in altered neuronal development. Our previous studies demonstrated cocaine inhibits neurite outgrowth in NGF-induced PC12 cells through dopamine, by activation of D₁ receptors. This study examined where cocaine interferes in the NGF signaling cascade. GSras1 cells that inducibly express activated forms of Ras upon treatment with dexamethasone were used. Morphological differentiation was quantified by counting cells bearing neurite-like processes after 72 h exposure to either dexamethasone or NGF alone, or with cocaine, dopamine or SKF-38393. Cocaine, dopamine, and the D₁ agonist inhibited neurite-like process outgrowth in both dexamethasone and NGF-induced GSras1 cells. GAP-43 expression, used as a measure for biochemical differentiation was severely diminished in NGF and dexamethasone-induced GSras1 cells treated with cocaine. These results suggest that cocaine, dopamine and activation of D₁ receptors affect the NGF signaling downstream, independent of ras expression, leading to altered neuronal differentiation.     

Gender- and region-specific expression of insulin receptor protein in mouse brain: effect of mild inhibition of oxidative phosphorylation

Summary. Insulin receptors (IR) and inhibition of oxidative metabolism have been suggested to partake in the pathophysiological cascade of neurodegenerative disorders. The goal of this study was to investigate gender- and region-specificity of insulin receptor protein expression in mouse brain subsequent to a mild hypoxic episode. Tissue was prepared from untreated male and female mice and animals pretreated in vivo with 20 mg/kg body weight i.p. 3-nitroproprionic acid (3-np; an inhibitor of succinic dehydrogenase) 1 hr prior to tissue preparation. IR expression in control animals was alike in males and females during proestrus and estrus but reduced during diestrus. On pretreatment, IR protein expression decrease in hippocampus in males but remained alike in other regions and females. In summary, IR protein expression is regionally different in males and females, gender-dependent, and modulated during the stages of the estrus cycle in females. Contrary to expectations it is not modified on mild inhibition of oxidative phosphorylation in any region in females and altered in hippocampus solely in males. The latter effect, however, warrants further scrutiny concerning participation in pathophysiological cascades affecting the hippocampus such as in Alzheimer’s disease. The first two authors contributed equally to this work.     

胰高血糖素样肽1受体激动剂在神经保护方面的研究进展

近年来的研究发现,与糖尿病并发的多种神经系统疾病,可随着治疗糖尿病的胰高血糖素样肽1受体激动剂(GLP-1RA)的使用而得到改善,但其具体机制尚不完全清楚。天然的GLP-1是进食诱导刺激回肠和结肠的L细胞分泌的肠肽类激素,其可促进胰岛素的合成和分泌。为克服GLP-1半衰期短而开发的多种长效GLP-1RA已经在临床上广泛应用,如艾塞那肽、利拉鲁肽、阿必鲁肽、度拉糖肽等,这些药物表现出在控制血糖水平和体重上的优势。由于GLP-1受体广泛分布于胰腺、肺、脑、心脏、肾和胃肠等组织细胞膜上,因此GLP-1RA的作用不仅仅在治疗糖尿病方面。已有的报道表明,GLP-1RA还具有显著的神经、心血管、肾脏保护,以及抗呼吸道炎症和减脂等多种作用。GLP-1受体在脑中亦有广泛分布,且GLP-1RA可有效通过脑血管屏障。GLP-1RA与相应受体结合,可激活PKA、PI3K/AKT、ERK、MEK等多个激酶信号通路,调节神经递质传递,这可能是GLP-1RA实现抗炎、减少氧化应激、抑制凋亡、减少DNA损伤、神经细胞修复,最终达到神经保护的途径。该文结合基础及临床研究,对GLP-1RA在缺血性脑卒中、认知功能障碍、...     

The role of microtubule-associated protein 1B in axonal growth and neuronal migration in the central nervous system

In this review,we discuss the role of microtubule-associated protein 1B(MAP1B) and its phosphorylation in axonal development and regeneration in the central nervous system.MAP1B exhibits similar functions during axonal development and regeneration.MAP1B and phosphorylated MAP1B in neurons and axons maintain a dynamic balance between cytoskeletal components,and regulate the stability and interaction of microtubules and actin to promote axonal growth,neural connectivity and regeneration in the central nervous system.     

pRb phosphorylation is regulated differentially by cyclin-dependent kinase (Cdk) 2 and Cdk4 in retinoic acid-induced neuronal differentiation of P19 cells

The retinoblastoma protein (pRb) is a key regulator of cell growth, differentiation and survival. pRb(-/-) mice show abnormal neuronal cell death in the developing brain. The function of pRb is regulated by its phosphorylation state. In this study, the phosphorylation of pRb during retinoic acid (RA)-induced neuronal differentiation of P19 cells was examined using site-specific antibodies against pRb phosphorylated at Ser601, Ser605 and Ser773. Although pRb was hyperphosphorylated in undifferentiated P19 cells, Ser601 and Ser773 were not phosphorylated. Upon exposure to RA, however, these two sites became strongly phosphorylated. Cdk4 kinase activity was almost undetectable in undifferentiated P19 cells, but was strongly activated on exposure to RA. In contrast, Cdk2 kinase activity and the phosphorylation of Ser605 were observed in undifferentiated cells as well as in RA-treated cells. These observations suggest that Cdk2 and Cdk4 may phosphorylate different sites of pRb in vivo and that the two sites of pRb examined here are newly phosphorylated during RA-induced neuronal differentiation in P19 cells.     

Characterization of NTera2/D1 cells as a model system for the investigation of cannabinoid function in human neurons and astrocytes

The limited availability and potential to culture primary human brain cells means that there is still a need for cell lines that reliably model human neurons and glial cells. The human-derived NTera2/D1 (NT2) cell line is a promising tool from which both neuronal (NT2N) and astrocytic (NT2A) cells can be derived in vitro. Here we have investigated the potential to use this cell model to investigate the endocannabinoid system in the CNS. Through immunocytochemical characterization with a range of neuronal and glial markers, we found that these cell lines differentiate into cells with immature neuronal and astrocytic phenotypes, respectively. By real-time PCR, immunocytochemistry, and functional inhibition of cAMP accumulation, the cannabinoid 1 receptors were identified only on NT2N cells, consistent with high levels of expression of this receptor in neuronal cells of the CNS. No evidence of cannabinoid 2 receptor expression was found on any of the NT2 cell types. Both the precursors and the differentiated NT2N and NT2A cells demonstrated mRNA expression for the key enzymes involved in endocannabinoid synthesis and degradation. This work establishes a cannabinergic phenotype in NT2N and NT2A cells, providing an alternative human derived renewable cell model for investigation of cannabinoid receptor function and endocannabinoid synthesis and metabolism in the CNS.     

Localization of protein kinase C theta immunoreactivity to interstitial cells of Cajal in guinea-pig gastrointestinal tract

In the gastrointestinal tract, interstitial cells of Cajal (ICC) are located between nerve fibres and muscle cells and have a role in neuromuscular transmission and muscle contractility. Protein kinase C (PKC) is involved in modulation of muscle contractility by neurotransmitters, but it is not known if PKC has a role in ICC. There are 11 different PKC isoforms. The presence of PKC isoforms in ICC in guinea-pig gastrointestinal tract was examined using fluorescence immunohistochemistry and confocal microscopy. Segments of guinea-pig stomach, duodenum, ileum, proximal and distal colon were fixed in zambonis fixative. Frozen sections and wholemounts were incubated with anti-PKC antibodies (alpha, beta, delta, epsilon, gamma, iota, lambda, mu, theta) followed by fluorescent secondary antibody. Only PKC theta (theta) immunoreactivity was found in ICC. None of the other PKC isoforms (alpha, beta, delta, epsilon, gamma, iota, lambda, mu) localized to the ICC. PKC theta immunoreactivity was prominent in ICC located between the circular and longitudinal muscle layers (ICC-MY) in all regions except stomach and within the circular muscle (ICC-IM) in the large intestine. PKC theta was not present in ICC in the deep muscular plexus in either duodenum or ileum. PKC theta immunoreactivity was present in the cell body and proximal processes of the ICC. The cells containing PKC theta also contained cKit confirming the cells were ICC. ICC-MY in the ileum also contained the neurokinin (NK) 1 receptor. In conclusion, PKC theta is present in pacemaker ICC, but its function is not yet known. Functional studies will be needed to determine the role of this kinase in ICC. Knowing the second messenger cascades and being able to manipulate subpopulations of ICC will add to our understanding of the molecular and cell biology of ICC networks within the gastrointestinal tract and may ultimately help in understanding the aetiology of some gastrointestinal motor pathologies.     

Ultrastructural localization of G-proteins and the channel protein TRP2 to microvilli of rat vomeronasal receptor cells

Microvilli of vomeronasal organ (VNO) sensory epithelium receptor cells project into the VNO lumen. This lumen is continuous with the outside environment. Therefore, the microvilli are believed to be the subcellular sites of VNO receptor cells that interact with incoming VNO-targeted odors, including pheromones. Candidate molecules, which are implicated in VNO signaling cascades, are shown to be present in VNO receptor cells. However, ultrastructural evidence that such molecules are localized within the microvilli is sparse. The present study provides firm evidence that immunoreactivity for several candidate VNO signaling molecules, notably the G-protein subunits G(ialpha2) and G(oalpha), and the transient receptor potential channel 2 (TRP2), is localized prominently and selectively in VNO receptor cell microvilli. Although G(ialpha2) and G(oalpha) are localized separately in the microvilli of two cell types that are otherwise indistinguishable in their apical and microvillar morphology, the microvilli of both cell types are TRP2(+). VNO topographical distinctions were also apparent. Centrally within the VNO sensory epithelium, the numbers of receptor cells with G(ialpha2)(+) and G(oalpha)(+) microvilli were equal. However, near the sensory/non-sensory border, cells with G(ialpha2)(+) microvilli predominated. Scattered ciliated cells in this transition zone resembled neither VNO nor main olfactory organ (MO) receptor cells and may represent the same ciliated cells as those found in the non-sensory part of the VNO. Thus, this study shows that, analogous to the cilia of MO receptor cells, microvilli of VNO receptor cells are enriched selectively in proteins involved putatively in signal transduction. This provides important support for the role of these molecules in VNO signaling.     

LRRN6A/LERN1 (leucine-rich repeat neuronal protein 1), a novel gene with enriched expression in limbic system and neocortex

Human chromosome 15q24-q26 is a very complex genomic region containing several blocks of segmental duplications to which susceptibility to anxiety disorders has been mapped (Gratacos et al., 2001, Cell, 106, 367-379; Pujana et al., 2001, Genome Res., 11, 98-111). Through an in silico gene content analysis of the 15q24-q26 region we have identifie1d a novel gene, LRRN6A (leucine-rich repeat neuronal 6A), and confirmed its location to the centromeric end of this complex region. LRRN6A encodes a transmembrane leucine-rich repeat protein, LERN1 (leucine-rich repeat neuronal protein 1), with similarity to proteins involved in axonal guidance and migration, nervous system development and regeneration processes. The identification of homologous genes to LRRN6A on chromosomes 9 and 19 and the orthologous genes in the mouse genome and other organisms suggests that LERN proteins constitute a novel subfamily of LRR (leucine-rich repeat)-containing proteins. The LRRN6A expression pattern is specific to the central nervous system, highly and broadly expressed during early stages of development and gradually restricted to forebrain structures as development proceeds. Expression level in adulthood is lower in general but remains stable and significantly enriched in the limbic system and cerebral cortex. Taken together, the confirmation of LRRN6A's expression profile, its predicted protein structure and its similarity to nervous system-expressed LRR proteins with essential roles in nervous system development and maintenance suggest that LRRN6A is a novel gene of relevance in the molecular and cellular neurobiology of vertebrates.     

The mRNA of transferrin is expressed in Schwann cells during their maturation and after nerve injury

Transferrin, the iron carrier protein, has been shown to be involved in oligodendroglial cell differentiation in the central nervous system but little is known about its role in the peripheral nervous system. In the present work, we have studied the presence of transferrin and of its mRNA in rat sciatic nerves and in Schwann cells isolated at embryonic and adult ages as well as during the regeneration process that follows nerve crush. We have also studied the correlation between the expression of the mRNAs of transferrin and the expression of mature myelin markers in the PNS. We show that transferrin is present in whole sciatic nerves at late stages of embryonic life as well as at postnatal day 4 and in adult rats. We demonstrate for the first time, that in normal conditions, the transferrin mRNA is expressed in Schwann cells isolated from sciatic nerves between embryonic days 14 and 18, being absent at later stages of development and in adult animals. In adult rats, 3 days after sciatic nerve crushing, the mRNA of transferrin is expressed in the injured nerve, but 7 days after injury its expression disappears. Transferrin protein in the sciatic nerve closely follows the expression of its mRNA indicating that under these circumstances, it appears to be locally synthesized. Transferrin in the PNS could have a dual role. During late embryonic ages it could be locally synthesized by differentiating Schwann cells, acting as a pro-differentiating factor. A similar situation would occur during the regeneration that follows Wallerian degeneration. In the adult animals on the other hand, Schwann cells could pick up transferrin from the circulation or/and from the axons, sub serving possible trophic actions closely related to myelin maintenance.     

Bone morphogenetic protein 4, inhibitor of differentiation 1, and epidermal growth factor receptor regulate the survival of cochlear sensory epithelial cells

Bone morphogenetic protein 4 (BMP4) is crucial for the development of the inner ear, but the mechanism of how BMP4 affects this process remains unknown. The focus of this research was to determine whether BMP4 can regulate the survival of cochlear sensory epithelial cells through inhibitor of differentiation 1 (Id1) and the epidermal growth factor receptor (EGFR). In this study, we first investigated the effects of BMP4, noggin, and dominant negative BMPR1B (dnBMPR1B) on the proliferation and survival of cochlear sensory epithelial cells. Subsequently, we investigated the influences of BMP4, noggin, and dnBMPR1B on the expression of Id1 and EGFR. We found that BMP4 had a negative effect on cell survival and on Id1 and EGFR expression in vitro, whereas noggin and dnBMPR1B treatment had positive effects. Knockdown of the Id1 gene with short interfering RNA (siRNA) reduced the expression of EGFR and cell proliferation. These data suggest that BMP4 may regulate survival through the Id1/EGFR pathway during development of the rat inner ear. © 2013 Wiley Periodicals, Inc.     

人His-AWP1融合蛋白表达载体的构建及其在原核生物的表达

目的 获取了His-AWP1融合蛋白，为了阶段深入研究AWP1的结构。功能及筛取与其相互作用的蛋白打下基础。方法 应用逆转录聚合酶链反应（RT-PCR）法从人ECV304内皮细胞系中克隆AWP1cDNA，并将其重组于能表达6个组氨酸残基的原核表达质粒pET-14b中，经酶切，序列鉴定，选择正确重组克隆。将其质粒转化大肠杆菌BL21（DE3），IPTG诱导表达，用Ni^2 -NTAHis柱纯化和SDS-PAGE分离蛋白。结果 克隆到一个627bp的AWP1cDNA片段，重组质粒目的DNA测序正确，纯化出了一个分子量约为38kD的融合蛋白，结论 用基因工程方法在原核细胞表达并成功纯化出His-AWP融合蛋白。     

Function of death‐associated protein 1 in proliferation,differentiation, and apoptosis of chicken satellite cells

Introduction: Muscle growth and regeneration are processes closely associated with proliferation, differentiation, and apoptosis of muscle cells. Death‐associated protein 1 (DAP1) has been identified as a negative regulator of autophagy. Little is known about the function of DAP1 in the regulation of myogenesis and satellite cells. Methods: Chicken satellite cells were transfected with DAP1 cloned into the pCMS–enhanced green fluorescent protein vector or pcDNA3.1 vector, or a small interference RNA against the endogenous DAP1 gene. The cells were assayed for proliferation, differentiation, and apoptosis. Results: The overexpression of DAP1 increased proliferation, differentiation, and myotube diameter, but it had no effect on satellite cell apoptosis. In contrast, knockdown of DAP1 significantly decreased proliferation, differentiation, and number of nuclei per myotube, and it increased apoptosis of the cells. Conclusion: DAP1 is required for regulating myogenesis and apoptosis of satellite cells, which may affect muscle mass accretion and regeneration, and ameliorate muscle sarcopenia. Muscle Nerve 48:777–790, 2013     

A putative tachykinin receptor in the cockroach brain: molecular cloning and analysis of expression by means of antisera to portions of the receptor protein

Tachykinins constitute a neuropeptide family that mediate their actions via a subfamily of structurally related G-protein-coupled receptors. Two receptors, Drosophila neurokinin receptor (NKD) and Drosophila tachykinin receptor (DTKR), with sequence similarities to mammalian tachykinin receptors have previously been cloned in Drosophila. In this study we have isolated a cockroach (Leucophaea maderae) cDNA clone by screening a brain cDNA library with a degenerate oligonucleotide probe based on a conserved sequence within the seventh transmembrane region of the Drosophila tachykinin receptors. This clone, Leucophaea tachykinin receptor (LTKR), encodes a portion of a putative receptor which could be aligned with the C-terminal half of members of the tachykinin receptor subfamily. In the fifth, sixth and seventh transmembrane regions the deduced amino acid sequence of LTKR exhibits 79% sequence identity to the DTKR receptor and 54% to that of NKD. This suggests that LTKR is orthologous to the DTKR receptor. To study the distribution of the predicted LTKR protein by immunocytochemistry, antisera were raised against synthetic peptides corresponding to a region of the third intracellular loop of LTKR. In the cockroach brain immunoreactive neuronal processes were seen in several synaptic neuropils of the protocerebrum and tritocerebrum as well as in the frontal ganglion. Some immunoreactive neuronal cell bodies were detected in the protocerebrum. Double labeling immunocytochemistry revealed that there is a substantial superposition between distribution of LTKR and processes containing tachykinin-related peptide (TRP). Some brain areas, however, only display TRP immunoreactive processes and no LTKR, suggesting the presence of at least one more TRP receptor type.     

gamma-Aminobutyric acid (GABA) signaling components in Drosophila: immunocytochemical localization of GABA(B) receptors in relation to the GABA(A) receptor subunit RDL and a vesicular GABA transporter

gamma-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in insects and is widely distributed in the central nervous system (CNS). GABA acts on ion channel receptors (GABA(A)R) for fast inhibitory transmission and on G-protein-coupled ones (GABA(B)R) for slow and modulatory action. We used immunocytochemistry to map GABA(B)R sites in the Drosophila CNS and compared the distribution with that of the GABA(A)R subunit RDL. To identify GABAergic synapses, we raised an antiserum to the vesicular GABA transporter (vGAT). For general GABA distribution, we utilized an antiserum to glutamic acid decarboxylase (GAD1) and a gad1-GAL4 to drive green fluorescent protein. GABA(B)R-immunoreactive (IR) punctates were seen in specific patterns in all major neuropils of the brain. Most abundant labeling was seen in the mushroom body calyces, ellipsoid body, optic lobe neuropils, and antennal lobes. The RDL distribution is very similar to that of GABA(B)R-IR punctates. However, the mushroom body lobes displayed RDL-IR but not GABA(B)R-IR material, and there were subtle differences in other areas. The vGAT antiserum labeled punctates in the same areas as the GABA(B)R and appeared to display presynaptic sites of GABAergic neurons. Various GAL4 drivers were used to analyze the relation between GABA(B)R distribution and identified neurons in adults and larvae. Our findings suggest that slow GABA transmission is very widespread in the Drosophila CNS and that fast RDL-mediated transmission generally occurs at the same sites.     

Effects of TNF-alpha and curcumin on the expression of thrombomodulin and endothelial protein C receptor in human endothelial cells

The objective of this study was to elucidate the effects of tumor necrosis factor-alpha (TNF-alpha) on the expression of thrombomodulin (TM) and endothelial protein C receptor (EPCR) in human endothelial cells as well as the effect of curcumin, a spice and coloring food compound, as a potential therapeutic agent. Human umbilical vein endothelial cells (HUVECs) treated with TNF-alpha (2.0 ng/ml) showed reduced TM mRNA levels by 80%, 97%, 94%, and 97% at 3, 6, 12, and 24 h, respectively (P<0.05), by real-time PCR analysis. Dose-dependent study showed that TM mRNA levels of HUVECs were decreased by 86%, 89%, 91%, and 94% after treatment of TNF-alpha (0, 0.25, 0.5, 1, and 2 ng/ml) for 6 h, respectively (P<0.05). TM protein levels in HUVECs were significantly reduced by 69% in TNF-alpha-treated cells as compared to controls (P<0.05) by Western blot analysis. Secreted protein and activity of TM of HUVEC cultures were also significantly reduced in TNF-alpha-treated cells. In addition, EPCR mRNA levels of HUVECs were significantly reduced in TNF-alpha-treated group as compared to controls (P<0.05). Furthermore, these effects were observed in other types of endothelial cells from human coronary arteries, lung, and skin. Curcumin effectively blocked these effects of TNF-alpha on downregulation of TM and EPCR. These data demonstrate that TNF-alpha significantly decreases expression of TM and EPCR at both mRNA and protein levels in several human endothelial cells. Curcumin can effectively block TNF-alpha-induced endothelial dysfunction. This study suggests a new molecular mechanism of inflammation-induced thrombosis and a new therapeutic strategy to prevent this clinical problem.